Blood pressure changes following chronic administration to rats of 3beta,16beta-dihydroxy-5-androsten-17-one, 3beta,17beta-dihydroxy-5-androsten-16-one and 21-hydroxy-4-pregnene-3,20-dione-21-acetate.

The urinary excretion of 3beta,16beta-dihydroxy-5-androsten-17-one (16beta-OH-DHEA) is increased in patients with low renin essential hypertension. This steroid and its isomer 3beta,17beta-dihydroxy-5-androsten-16-one (16-oxo-A) have also been reported to have mineralocorticoid activity in adrenalectomized rats. These findings have led to the postulate that excessive secretion of 16beta-OH-DHEA may be responsible for the production of low renin essential hypertension. In this study unilaterally nephrectomized salt loaded rats injected once a week with 30 mg of 11-desoxycorticosterone acetate per/kg of body weight for 2 month periods developed hypertension. Rats given similar amounts of 16beta-OH-DHEA or 16-oxo-A and rats given no steroids did not develop hypertension. We conclude that it is unlikely that 16beta-OH-DHEA and 16-oxo-A are direct causative factors in the production of low renin essential hypertension.     

Induction of vaginal mucification in rats with testosterone and 17beta-hydroxy-5alpha-androstan-3-one.

Based on histological criteria, Kingsley and Bogdanove (3) reported that the benzoate ester of 17beta-hydroxy-5alpha-androstan-3-one (5alpha-DHT), unlike testosterone propionate, is unable to induce vaginal mucification when given subcutaneously to rats. In contrats, Kennedy (4) found in estrogen-pretreated rats that both 5alpha-DHT and testosterone induced vaginal mucification as indicated by increased vaginal sialic acid concentration.To determine if esterification of these androgens altered their ability to induce vaginal mucification, ovariectomized rats, pretreated for 3 days with 0.25 mug estradiol-17beta, were treated for 8 days with either sesame oil or 7 mumoles of testosterone, 5alpha-DHT and their respective propionate and benzoate esters. All treatments except 5-alpha-DHT benzoate increased vaginal weight and vaginal mucification, as assessed histochemically and biochemically. 5alpha-DHT propionate was less effective than 5alpha-DHT while testosterone benzoate, but not propionate was less effective than testosterone. To determine if estrogens are necessary for the vaginal effects of androgens, ovariectomized and ovariectomized-adrenalectomized rats were treated with testosterone or 5alpha-DHT. Adrenalectomy did not significantly affect the vaginal response to either androgen. It is therefore concluded that androgens are capable of inducing vaginal mucification in the absence of estrogens.     

Synthesis of the allylic gonadal steroids, 3 alpha-hydroxy-4-pregnen-20-one and 3 alpha-hydroxy-4-androsten-17-one, and of 3 alpha-hydroxy-5 alpha-pregnan-20-one

A method for the convenient synthesis of the recently isolated allylic gonadal steroids, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha-dihydroprogesterone; 3 alpha-DHP) and 3 alpha-hydroxy-4-androsten-17-one (3 alpha-HA), was developed using 4-pregnene-3,20-dione (progesterone) and 4-androstene-3,17-dione as substrates and potassium trisiamylborohydride (KS-Selectride) as reducing agent. Similar reactions were also used for the reduction of 5 alpha-pregnane-3,20-dione to 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-HP). The yields were about 15%, 50%, and greater than 90% for 3 alpha-DHP, 3 alpha-HA and 3 alpha-HP, respectively. Structures of the products, including the 3 beta-isomers and the 17 alpha-epimer, formed in these reactions were determined by NMR and mass spectroscopic methods.     

Synthesis of 16 alpha,19-dihydroxy-4-androstene-3,17-dione and 3 beta,16 alpha,19-trihydroxy-5-androsten-17-one and their 17 beta-hydroxy-16-keto isomers

3 beta,16 alpha,19-Trihydroxy-5-androsten-17-one and 16 alpha,17-dihydroxy-4-androstene-3,17-dione were synthesized from the 5 alpha-bromo-6 beta,19-epoxy-17-ketone derivative 1, using the bromination at C-16 alpha of the 17-ketone 1 and the controlled alkaline hydrolysis of the 16 alpha-bromo-17-ketones 2 and 11 as key reactions. Zinc dust reductive cleavage of the 6 beta,19-epoxy-16 alpha-hydroxy-17-ketones 4 and 12, produced by controlled hydrolysis, gave the corresponding 19-alcohol derivatives 6 and 14, which were rearranged to the 17 beta-hydroxy-16-ketones 7 and 15 when treated with sodium hydroxide. The 3 beta,16 alpha,17 beta,19-tetrol 8 was obtained from the 16 alpha-ketol 6 by reaction with sodium borohydride.     

Formation of 5-[17beta-2H]androstene-3beta,17alpha-diol from 3beta-hydroxy-5-[17,21,21,21-2H]pregnen-20-one by the microsomal fraction of boar testis

After incubation of 3beta-hydroxy-5-[17,21,21,21-2H]-pregnen-20-one with the microsomal fraction of boar testis, the metabolites were analyzed by gas chromatography and gas chromatography-mass spectrometry. The following metabolites were identified: 3beta,17alpha-dihydroxy-5-[21,21,21-3H]pregnen-20-one, 3beta-hydroxy-5-androsten-17-one, 5-androstene-3beta,17beta-diol, and 5-[17beta-2H]androstene-3beta,17alpha-diol. The presence of a 2H atom at the 17beta position of 5-androstene-3beta,17alpha-diol was confirmed by oxidizing the steroid with 3beta-hydroxy-steroid dehydrogenase of Pseudomonas testosteroni to obtain 17alpha-hydroxy-4-[2H]androsten-3-one and then by oxidizing the latter steroid with chromic acid to obtain nonlabeled 4-androstene-3,17-dione. Among these metabolites, the first three can be interpreted to be synthesized by a well documented pathway, including 17alpha-hydroxylation followed by side chain cleavage as follows: 3beta-hydroxy-5-[17,21,21,21-2H]pregnen-20-one leads to 3beta,17alpha-dihydroxy-2-[21,21,212H]-pregnen-20-one leads to 3beta-hydroxy-5-androsten-17-one leads to 5-androstene-3beta,17beta-diol. On the other hand, 5-androstene-3beta,17alpha-diol, which contained a 2H atom at the 17beta position, is not likely to be synthesized via above mentioned pathway in which nonlabeled 3beta-hydroxy-5-androsten-17-one is formed as the first C19-steroid. It seems that an alternate side chain cleavage mechanism leading from pregnenolone to 17alpha-hydroxy-C19-steroid exists in boar testis.     

Metabolism of 5 alpha-androstane-3 beta,17 beta-diol to 17 beta-hydroxy-5 alpha-androstan-3-one and 5 alpha-androstan-3 alpha,17 beta-diol in the rat.

Significant metabolism of 5 alpha-androstane-3 beta,17 beta-diol to 17 beta-hydroxy-5 alpha-androstan-3-one was recorded in several tissues and organs from rats and humans. This bioconversion was further investigated in rat testis homogenates. 5 alpha-Androstane-3 beta,17 beta-diol was readily metabolized to 17 beta-hydroxy-5 alpha-androstan-3-one with NAD and/or NADP added as cofactors. When a NADPH generating system was included in the incubation, 5 alpha-androstane-3 beta,17 beta-diol was metabolized to 5 alpha-androstan-3 alpha,17 beta-diol. Only small amounts of 17 beta-hydroxy-5 alpha-androstan-3-one accumulated under the latter condition.     

Inhibitor specificity of the placental microsomal oxidase system responsible for the aromatization of epitestosterone (17 alpha-hydroxy-4-androsten-3-one)

Human placental microsomes converted epitestosterone to estradiol-17 alpha at rates of 23-48 pmol/min X mg protein with a Km of 113 microM. Activity was inhibited 70-90% by concentrations of CO, metyrapone, n-octylamine, 7,8-benzoflavone and 7-ethoxycoumarin which had no effect on the aromatization of 4-androstene-3, 17-dione. Conversely, cyanide and azide were more effective inhibitors of the conversion of the latter androgen. A variety of neutral steroids inhibited the aromatization of epitestosterone with 19-norsteroids being particularly effective, but competitive effects could not be demonstrated. Both 17 beta-hydroxy-4-estren-3-one and 16 alpha-hydroxy-4-androstene-3,17-dione caused a mixed inhibition. A number of phenolic steroids were also inhibitory with 16-oxo compounds being particularly effective. Inhibition by estrone was non-competitive (Ki = 16 microM). The aromatization of epitestosterone resembles placental microsomal oxidase activities against estrone and benzo [a]pyrene in its inhibitor specificity and epitestosterone may be the native substrate for an oxidase also active in the metabolism of aromatic xenobiotic chemicals.     

Purification and properties of testicular 3 beta-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase

Through the treatment of rat testicular microsomes with sodium cholate, 3 beta-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase (abbreviated as the 3 beta-hydroxysteroid dehydrogenase and isomerase, respectively) were solubilized, and then purified by DEAE and hydroxylapatite column chromatographies. The findings were as follows: With this purification procedure, the 3 beta-hydroxysteroid dehydrogenase activity could not be separated from the isomerase. For 3-oxo-4-ene-steroid formation from 3 beta-hydroxy-5-ene-steroids, NAD+ was required as a cofactor. While the 3 beta-hydroxysteroid dehydrogenase required NAD+, the isomerase also required NAD+ or its reduced form, in contrast to the microbial enzyme. On treatment of the purified enzyme with 5'-p-fluorosulfonyl-benzoyladenosine (FSBA), both enzyme activities were markedly reduced. The enzyme, affinity labeled with [adenine-8-14C]FSBA, showed a mol. wt of 46.8 K. During 4-androstenedione production from DHA, 5-androstenedione was detected as an intermediate.     

An approach to the mechanism of the potent antiglucocorticoid: 17 beta-hydroxy-11 beta-4-dimethylaminophenyl-17 alpha-propynyl-estra-4,9-dien-3-one

The effects of a new synthetic steroid, 17 beta-hydroxy-11 beta-4-dimethyl-aminophenyl-17 alpha-propynyl-estra-4,9-dien-3-one, classified under reference R38486 in the Roussel-Uclaf nomenclature [1], were investigated in two established rat hepatoma cell lines in order to gain information on the mechanism of action. The induction of tyrosine aminotransferase (TAT) and alanine aminotransferase (AAT) was totally abolished when R38486 was added with dexamethasone either on a 1-1 basis or on a 10-fold excess depending on the differentiation state of the cell. Binding studies showed a high affinity for the glucocorticoid receptor; however our "whole cell" study with [3H] R38486 indicates that only a low amount of antagonist-receptor complexes was translocated into the nucleus. Nuclear fractionation experiments showed that R38486, like the other antagonists studied, was located in the chromatin fraction where it may exert some definite role. Our observations based on whole cell experiments using physiological doses of glucocorticoid analogs indicate that the binding of activated antiglucocorticoid-receptor complexes to nuclear acceptor sites represents a physiologically significant process. Moreover the differences in the nuclear binding of antagonist-receptor- as compared to agonist-receptor-complexes may set off the machinery of antagonistic action.     

Testosterone, 17 beta-hydroxy-5 alpha-androstan-3-one and 4-androstene-3, 17-dione in the plasma of male and female grey squirrels (Sciurus carolinensis)

Extracts of squirrel plasma have been chromatographed on partition columns (using a hydrophilic stationary phase) at atmospheric pressure (Celite support) and a reversed phase system at high pressure (HPLC). Both methods effectively separated testosterone, 17 beta-hydroxy-5 alpha-androstan-3-one (DHT) and 4-androstene-3,17-dione; they gave elution patterns that differed considerably. Chromatographic mobility of the three androgens on the two systems was identical with that of fractions of squirrel plasma extracts that gave responses measured by appropriate androgen radioimmunoassays; good evidence for the occurrence of these androgens in squirrel plasma is thus provided. Plasma testosterone levels were 300 pmol/l in juvenile males, 800-7000 pmol/l in sexually-active males but undetectable (less than 50 pmol/l) in sexually-regressed males. Plasma DHT levels were also high in sexually-active males, but undetectable in other males except for one regressed individual. Plasma androstenedione was higher in juvenile males than in adult males, in which it was similar whether or not they were sexually regressed. Plasma testosterone and DHT, unlike androstenedione, were totally dependent on the presence of the testes. In females testosterone and DHT were undetectable in plasma but androstenedione levels were high, especially at oestrus. Androstenedione was dependent on the presence of the ovaries.